SQ109 is a new anti-TB drug candidate that is currently undergoing Phase Ia Clinical Trials in humans. SQ109 was selected out of the library of 63,238 compounds, because it is highly efficacious in killing M. tuberculosis within infected macrophages and in experimental animals, has relatively low cytotoxicity in cultured mammalian cells, has activity against both drug-susceptible and drug-resistant M. tuberculosis, and has drug-like pharmacokinetic and pharmacodynamic profiles. Any new anti-TB drug will be used in combination with other drugs in order to prevent the emergence of drug resistant M. tuberculosis. How SQ109, a new drug compound with unique attributes, interacts with existing first line anti-TB drugs is crucial for us to understand, as this knowledge enables us to design the most efficacious therapeutic combination and to avoid any antagonistic side effects of a new combination therapy. We investigated the drug interaction in various combinations of SQ109 with the first line anti-TB drugs in both in vitro assays and in vivo murine models of TB. We found that SQ109 preferentially synergized with RIF in in vitro growth inhibition assays and demonstrated enhanced bactericidal activity in mice when used in combination with INH and RIF. These findings are significant for optimizing the drug combination therapies to be tested in future clinical trials. In this exploratory R21 grant Application we will: 1. Assess whether SQ109 is metabolized within M. tuberculosis and how co-administration of RIF influences SQ109 metabolism. If SQ109 metabolites exist, we will study their structures by mass spectrometry and will compare them to the SQ109 metabolites generated by human CYP. If the metabolites are the same, we will prepare and purify them in sufficient quantities and test them against M. tuberculosis for potency in vitro. 2. Determine the expression of individual CYP in RIF-treated M. tuberculosis in order to identify candidate SQ109 activators that are induced by RIF. 3. Generate M. tuberculosis knock out mutants of the candidate CYP(s) and re-examine the synergy of interaction between SQ109 and RIF in the CYP knockout mutants. Public Health Relevance: One of the most vexing problems with standard tuberculosis (TB) therapy is the lengthy treatment course (6-9 months) and toxicity of the drugs in the regiment. This prolonged therapeutic regimen results in a high level of noncompliance, and ineffective concentrations of drugs lead to the emergence of multidrug resistant (MDR) M. tuberculosis. There are an estimated 400,000 new cases of MDR TB each year, and the World Health Organization (WHO) estimates there are 50 million individuals worldwide infected with a resistant strain of TB. MDR strains of M. tuberculosis cause increased mortality, and the fatality rate of untreated cases of MDR TB can be as high as 70%. The likelihood of cure for MDR M. tuberculosis-infected individuals becomes even less in the setting of secondary immunosuppression caused by HIV and malnutrition, conditions that are rampant in developing countries. Because of the failures of the existing therapies, the world desperately needs new drugs that can shorten treatment duration and kill MDR M. tuberculosis. Sequella, Inc., founded in 1997, focuses its R&D on developing new diagnostic tools and new drugs for TB disease. One of our most successful projects is the development of SQ109, a new anti-TB drug candidate that is currently undergoing Phase Ia Clinical Trials. SQ109 was selected as our lead drug candidate out of the library of 63,238 compounds, because it is highly efficacious in killing M. tuberculosis within infected macrophages and in experimental animals, has relatively low cytotoxicity in cultured mammalian cells, has activity against both drug-susceptible and drug-resistant M. tuberculosis, and has drug-like pharmacokinetic and pharmacodynamic profiles. Importantly, when used in combination with rifampin (RIF) + isoniazid (INH) + pyrazinamide (PZA), SQ109 is able to increase bacterial killing and reduce the time by at least 25% to achieve the same level of CFU as the standard drug regimen, RIF + INH + PZA + ethambutol (EMB) in a mouse model of TB. Any new anti-TB drug will be used in combination with other drugs in order to prevent the emergence of drug resistant M. tuberculosis. How SQ109, a new drug compound with unique attributes, interacts with existing first line anti-TB drugs is crucial for us to understand, as this knowledge enables us to design the most efficacious therapeutic combination and to avoid any antagonistic side effects of a new combination therapy. In this R21 exploratory grant application, we propose to investigate one of the several hypotheses that may contribute to the synergistic interaction between SQ109 and RIF in M. tuberculosis: (1) SQ109 is readily metabolized in M. tuberculosis, (2) RIF is able to induce the expression of one or more M. tuberculosis CYP- like proteins, and (3) CYP induced by RIF is involved in SQ109 metabolism. Tasks 1 and 2 are two independent areas of study, which are linked by task 3. In this application, we outline the experiment design for all three tasks in sequence. Further, we provide alternative directions for the study of this interesting synergy if the data obtained do not support our hypothesis.